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International Journal of Bioprinting Control nutrients to manipulate fungal growth

was particularly visible after 4 days of growth and was 3.5. Local variations in nutrients in a 3D complex
still apparent after 10 days, albeit less strongly. On the structure guides the growth and foraging behavior
contrary, when the zone had a high nutrient level (Design of mycelium
II and Design IV), both extension and bridging were Since ELMs exist in various shapes and complexities,
promoted and the density of mycelium over this zone the effects of varying nutrients on all three axes should
became indistinguishable from the rest of the network also be investigated. Furthermore, 3D printing via DIW
on the ELM. It should be noted, nevertheless, that the conveniently allows for 3D shapes to be built and the
extension and bridging are mostly occurring after 10 inks were formulated to allow both extrudability and
days. When comparing designs containing high-nutrient buildability. The extension and growth of the mycelium
zones (Design II and Design IV) to designs containing from the two species could therefore be observed in three
low-nutrient zones (Design I and Design III), the mycelia dimensions and in complex patterns (Figure 5).
emerging from zones of origin were denser for designs
containing high-nutrient zones, presumably due to the To test the growth of mycelium in the vertical direction,
diffusion of malt and peptone from high-nutrient zones cuboid structures were designed where the base of the
to the zones of origin. For designs with low-nutrient cube is the zone of origin (Figure 5A). The structures were
zones, the opposite may also be true, where the initial again 3D-printed with pores to allow air to flow through
mycelium formed was sparser due to the diffusion of malt the structure and homogeneous growth at the core of the
and peptone from the zones of origin to the low-nutrient sample. After the base was printed, the subsequent layers
zones. were then printed using inks with either a low- or high-
nutrient content. For both strains of fungi, the mycelium
Similar observations were made for the same designs first emerged from the base of the structure 4 days after
containing G. lucidum for their extension and bridging in printing. However, at this point, the network was barely
the four multi-material structures (Figure 4C). While the visible, containing only a few strands of hyphae. By day 7,
behavior resembled that of P. ostreatus, the mycelium of G. the mycelium became more visible as the subsequent layers
lucidum had the tendency to conform to the underlying above were gradually covered until complete coverage
structure with less likelihood to spread its hyphae in all observed by day 10. If the nutrient content of the higher
directions, resulting in the shape profile of the underlying layers is low, the mycelium formed in the upper layers is
structure being more pronounced (compared with sparse, and vice versa. The designs also demonstrated
Figure 1D). Meanwhile, for P. ostreatus, there was also a that not all inks used in the fabrication process need to
greater tendency for the mycelium to extend beyond the include liquid mycelium. Indeed, only a section needs to
3D-printed structure and onto the Petri dish housing the be inoculated and the mycelium will eventually envelop the
ELM. Otherwise, the growth behavior of both G. lucidum entire structure. This is interesting as it potentially reduces
and P. ostreatus were consistent with the phalanx/guerrilla the amount of liquid mycelium required, which should be
theory on foraging behavior. easier for the fabrication of large-volume structures. It also
Both Designs I and II also showed the potential of facilitates the ink preparation and storage as the ink without
varying local nutrient contents to either promote or inhibit mycelium can be stored in the fridge until use, whereas
the self-healing capabilities of fungal mycelium. It has been inks containing mycelium need to be prepared fresh before
established that live mycelium materials can regenerate, use. If an ink containing mycelium is kept for too long,
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allowing holes on mycelium sheets to be filled or two mycelium may develop inside any air bubbles present
separate structures to combine when placed together. within the ink, ultimately compromising the homogeneity
By placing a material of low-nutrient content between of the ink leading to 3D printing difficulties later.
two regions containing fungal mycelium, the bridging of From the preceding observations, it has been established
mycelium between the two region was inhibited and the that mycelium growth can be controlled through the
self-healing property of the fungal mycelium could be precise deposition of nutrients in the substrate, allowing for
suppressed to a certain extent. Meanwhile, the self-healing structures with various surface patterns to be fabricated (see
capability of mycelium could be promoted if the material also Figures S9 and S10, Supporting Information). As such,
in between contained high nutrient levels, promoting the a potential application of this technology is the creation of
growth of mycelium over the gap. As such, this study also distinct and detailed patterns on the surface of structures.
showed the possibility of controlling this phenomenon in Positioning zones of high- and low-nutrient contents in
fungal-based ELMs. the ELM should guide the growth of mycelium in specific
Therefore, the growth behavior of the two fungi on directions, allowing for the fabrication of structures with
a flat 2D plane was observed and their behavior in a 3D esthetical value. Figure 5B highlights an example of this
space was then studied. application. Using a low-nutrient zone, the growth of a

Volume 10 Issue 5 (2024) 177 doi: 10.36922/ijb.3939